Yersinia pestis is a Category A Select Agent capable of causing highly fatal, highly contagious pneumonic plague. At present, there is no licensed vaccine for plague, creating an enormous need to rely on currently available antibiotics for treatment and post-exposure prophylaxis. Recent emergence of multi-antibiotic resistant Y. pestis underscores the importance of minimizing the use of antibiotic resistance cassettes for research purposes in order to maximize the availability of treatment options for human plague. However in so doing, one compromises the potential for genetics in Yersinia pestis as currently available research tools to genetically manipulate the bacteria all involve the introduction of antibiotic resistance at one or more steps. We seek to improve the availability of genetic tools to study the pathogenesis of Yersinia pestis through the creation of novel plasmids which involve selection based on metabolic pathways rather than antibiotic resistance. Plasmid borne resistance to divalent metal ions, for example mercury or copper, has been studied in both Gram negative and Gram positive bacteria and is well characterized. In addition, nutrient auxotrophy, such as the diaminopimelic acid biosynthetic pathway, can be exploited as a means to select plasmid DNA both in vivo and in laboratory media. The utility of these pathways for genetic tools such as plasmids and suicide vectors will be explored. These tools will then be employed to create specific mutations in Yersinia pestis and studied in the context of cell culture and animal infections. [unreadable] [unreadable] [unreadable]